Infection by protozoan parasites causes some of the most prevalent and devastating diseases worldwide. With a sparse yet dwindling arsenal of useful drugs and a paucity of effective vaccines, identifying new targets for therapeutic intervention has become imperative. Despite their enormous diversity, many protozoan parasites rely upon a common strategy centered on a repertoire of transmembrane adhesins to gain close contact with host cells. Severing enzymes are then often deployed to modulate these initial interactions. At the core of this strategy are parasite-encoded rhomboid proteases that cleave many different families of adhesins in diverse intracellular and extracellular parasites. Recent advances in genetic manipulation have validated these enzymes as promising therapeutic targets for remarkably diverse parasitic diseases. Yet the promise of this exciting progress rests on deciphering the enzymatic mechanism of rhomboid proteases at sufficient clarity to guide therapeutic targeting. Rhomboid proteases pose a challenge to existing enzyme paradigms: their conserved six transmembrane segments fold into a helical bundle inside the cell membrane with a novel protease active site at its core. Understanding the principles underlying such membrane-immersed catalysis is lacking due to a paucity of tools with which to study it in quantitative and unbiased ways. We recently developed the first quantitative assays for interrogating rhomboid architectural stability, reaction kinetics directly within the membrane, and transmembrane protein dynamics using spectroscopic methods. Our preliminary studies suggest an unanticipated new mechanism of action for membrane-immersed proteolysis, a means for it's direct regulation by cellular ions, and directly observed the first catalytic comple between enzyme and substrate. Capitalizing on these advances, we propose to leverage our newly developed methods to delineate rhomboid enzyme mechanism in quantitative terms. The outcome of these lines of investigation have direct and immediate implication on targeting these key enzymes for the treatment of various parasitic diseases that kill millions of people worldwide.